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doi:10.1038/nature11542
Supplementary Discussion
Transport mechanism
Combination of the crystal structure and transport and binding assays of the vcINDY presented
here, along with previous biochemical characterization of its bacterial, fly and, in particular,
mammalian homologs 1-4, suggest a transport mechanism for the Na+-dependent dicarboxylate
transporter from Vibrio cholerae (Supplementary Fig. 17). In the “substrate-waiting,” outwardfacing Co conformation, the transporter adopts a structure in which its N-terminal half is in the
“U-shape” while the C-terminal half is in the “V-shape”, N(U)-C(V), with its Na+- and substratebinding sites facing the extracellular space. Following the induction of the proper substrate
pocket by the binding of Na+ ions, probably two, a substrate molecule binds to the transporter,
yielding the Co-S-Na+ state. Using the binding energy and aided by Brownian motion, the
transporter converts to the Ci-S-Na+ state. In this state, the palm of the N-terminal half moves
toward the cytosol, whereas its thumb stays at its original position in the membrane, yielding an
inverted V-shape. At the same time, the C-terminal half of the transporter changes from the Vshape to a U-shape. In this N(V)-C(U) structure, the substrate is exposed to the cytosolic space.
Substrate release to the cytosol is then triggered by the escape of the Na2 ion, and is followed by
the release of the Na1 ion. Finally, the transporter returns from this Ci state back to the Co state.
Several pieces of published experimental data on various INDY proteins support the above
transport mechanism. Transport kinetics studies on both the rabbit and the human NaDC1
support a binding order of three Na+ ions from the extracellular space, followed by dicarboxylate
5,6
. Similar observations have been made for the sequence of binding of the two Na+ ions and one
substrate for bacterial INDY homologs 7-9. Accessibility studies of cysteine substitution in
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mammalian NaDC1 using a membrane-impermeable sulfonate reagent have shown that the tip of
HPout, including its SNT motif, is accessible from the extracellular space 10-12. Such accessibility
of the hairpin tip is increased in the presence of Na+ ions, but access to the cysteine labeling is
protected by the addition of substrate 11,12. The equivalent of another segment near the substrate
binding site, Ser200 – Pro202 in vcINDY, has also been found to be accessible from the
extracellular side in the human sulfate transporter NaS1 13. All of the above evidence supports an
induced-fit, alternating access model of Na+-dicarboxylate co-transport 6,14.
For the vcINDY dimer, this transport mechanism implies that the two clusters of helices at the
protomer-protomer interface (TMs 2, 3, 7 and 8 from both protomers) form an anchoring
scaffold for the transporter in the membrane (Fig. 1d and Supplementary Fig. 17). Connected by
TM4a and TM9a as their respective hinges, the two helical bundles in the N- and C-terminal
hands move up and down relative to the anchoring scaffold in opposite directions along the
membrane normal (Supplementary Fig. 9), realizing an alternating access model of substrate
translocation across the membrane.
As the bound citrate in our vcINDY structure is exposed to the cytosolic space, the structure
represents the transporter’s inward-facing, substrate-bound conformation, Ci-S-Na+ 14. This
interpretation is also in agreement with our observations that citrate interacts with the transporter
in solution but inhibits its transport activity only slightly from outside of the cell (Fig. 1b and
Supplementary Fig. 5). It follows that citrate is a Ci-conformation specific inhibitor. As an
importer, the low energy state of vcINDY in the absence of substrate should be the outwardfacing conformation, Co. Following Na+ and substrate binding, the transporter converts from the
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Co-S-Na+ state to the Ci-S-Na+ state for cytosolic release. In the present work, however, because
a high concentration of citrate was present in the crystallization buffer, a pre-release Ci state of
the transporter was captured in the crystal structure.
The vcINDY structure reported here represents its inward-facing, Ci-S-Na+ conformation, a prerelease state (Supplementary Fig. 17). As the protein functions as an importer, its substrate
selectivity depends on the atomic structure of the substrate-binding site in its outward-facing Co
or Co-Na+ conformation, a pre-binding state. Between the outward- and inward-facing states, the
binding sites for Na+ ions are likely to be conserved. The interactions between the two SNT
motifs and the two carboxyl groups at the ends of the substrate molecule are also likely to be
conserved. Additional residues at the binding site that interact with the substitutions at the
middle carbons probably provide the fine tuning of the substrate specificity of the transporter 9
that, for example, allows it to distinguish between different kinds of dicarboxylate molecules.
Substrate Specificity of sulfate transporters in the SLC13 family
The substrate-binding pocket in our crystal structure also explains the substrate preference
between carboxylate transporters and sulfate transporters among SLC13 proteins 2-4. In the
sulfate transporters the substrate probably binds at the same location. At the third residue of the
C-terminal SNT motif, in both NaS1 and NaS2, there is a proline residue there instead of a
threonine or a valine as found in the carboxylate transporters (Fig. 4c, Supplementary Fig. 3).
Interestingly, when we mutated Thr379 in vcINDY into a proline, the transport of succinate
became sensitive to sulfate competition (Fig. 4d, Supplementary Fig. 15). In addition, there is a
conserved serine residue at the vcINDY-P201 position among sulfate transporters. Previous
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studies have shown that a substitution of the serine by alanine in the human NaS1 resulted in an
increased Km for sulfate transport 13.
Comparison with CNT
Although the amino acid sequence varies, the structure of the hairpin tip – capping loop motif is
conserved between the two proteins, suggesting that this structural motif may be a common Na+binding motif in membrane transporter proteins. While vcINDY and CNT share a similar core
structure, the ways by which each core is linked to its respective scaffold are completely
different. CNT forms a trimer, with three pairs of long helices forming a scaffold around the
threefold axis, which links to the core from each protomer (Supplementary Fig. 16). In contrast,
in the dimeric vcINDY, the two palms in the core are each connected by a hinge to the anchoring
thumbs, which cluster around the center of the dimer. The helical bundle of the palm forms a
relatively well-defined structure for Na+ binding, with the substrate sitting between the two
oppositely-inverted palms. While the flexible linker between the palm and the thumb allows the
two hands to change shape in a symmetrical manner 15, the anchoring scaffold formed by the
thumbs at the center of the dimer allows the N- and C-terminal palms to move up and down in
opposite directions in the membrane to propel substrate translocation. Such domain movement
relative to an anchor scaffold during substrate translocation has been observed previously in the
trimeric Na+-dependent glutamate transporter 16.
References
1
2
Wright, E.M., Transport of carboxylic-acids by renal membrane-vesicles. Ann Rev
Physiol 47, 127-141 (1985).
Pajor, A.M., Sodium-coupled transporters for Krebs cycle intermediates. Ann Rev Physiol
61, 663-682 (1999).
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3
4
5
6
7
8
9
10
11
12
13
14
15
16
Markovich, D. & Murer, H., The SLC13 gene family of sodium sulphate/carboxylate
cotransporters. Pflug Arch Eur J Phy 447, 594-602 (2004).
Pajor, A.M., Molecular properties of the SLC13 family of dicarboxylate and sulfate
transporters. Pflug Arch Eur J Phy 451, 597-605 (2006).
Wright, S.H., Hirayama, B., Kaunitz, J.D., Kippen, I., & Wright, E.M., Kinetics of
sodium succinate cotransport across renal brush-border membranes. J Biol Chem 258,
5456-5462 (1983).
Yao, X. & Pajor, A.M., The transport properties of the human renal Na+-dicarboxylate
cotransporter under voltage-clamp conditions. Am J Physiol Renal Physiol 279, F54-64
(2000).
Hall, J.A. & Pajor, A.M., Functional characterization of a Na+-coupled dicarboxylate
carrier protein from Staphylococcus aureus. J Bacteriol 187, 5189-5194 (2005).
Hall, J.A. & Pajor, A.M., Functional reconstitution of SdcS, a Na+-coupled dicarboxylate
carrier protein from Staphylococcus aureus. J Bacteriol 189, 880-885 (2007).
Strickler, M.A., Hall, J.A., Gaiko, O., & Pajor, A.M., Functional characterization of a
Na+-coupled dicarboxylate transporter from Bacillus licheniformis. Bba-Biomembranes
1788, 2489-2496 (2009).
Pajor, A.M., Krajewski, S.J., Sun, N., & Gangula, R., Cysteine residues in the
Na+/dicarboxylate co-transporter, NaDC-1. Biochem J 344, 205-209 (1999).
Pajor, A.M., Conformationally sensitive residues in transmembrane domain 9 of the
Na+/dicarboxylate co-transporter. J Biol Chem 276, 29961-29968 (2001).
Pajor, A.M. & Randolph, K.M., Conformationally sensitive residues in extracellular loop
5 of the Na+/dicarboxylate co-transporter. J Biol Chem 280, 18728-18735 (2005).
Li, H. & Pajor, A.M., Serines 260 and 288 are involved in sulfate transport by hNaSi-1. J
Biol Chem 278, 37204-37212 (2003).
Law, C.J., Maloney, P.C., & Wang, D.N., Ins and outs of major facilitator superfamily
antiporters. Annu Rev Microbiol 62, 289-305 (2008).
Forrest, L.R. & Rudnick, G., The rocking bundle: a mechanism for ion-coupled solute
flux by symmetrical transporters. Physiology (Bethesda, Md 24, 377-386 (2009).
Reyes, N., Ginter, C., & Boudker, O., Transport mechanism of a bacterial homologue of
glutamate transporters. Nature 462, 880-885 (2009).
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Supplementary Table 1. Crystallographic data collection and reduction statistics for phasing
Data set
1
Unit-cell
parameters
a, b, c (Å)
β (°)
2
3
4
All (1-4)
101.42, 101.41, 101.56, 101.64, 101.73, 101.04, 102.83, 100.85, 101.88, 101.24,
165.12
165.73
165.50
165.74
165.53
101.96
102.12
101.58
100.78
101.61
Number of
frames
360
360
360
360
1,440
Bragg spacings
(Å)
40-3.5
(3.59-3.5)
40-3.7
(3.8-3.7)
40-4.0
(4.1-4.0)
40-4.0
(4.1-4.0)
40-3.5
(3.59-3.5)
Measurements
313,099
258,201
213,160
199,285
1,002,530
Unique
reflections
41,571
34,565
28,103
28,068
41,621
Multiplicity
7.5 (7.3)
7.5 (6.2)
7.6 (7.4)
7.1 (7.4)
24.1 (7.4)
Completeness
(%)
99.3 (93.2)
98.2 (74.9)
99.8 (100.0)
99.6 (100.0)
99.4 (94.1)
Rmerge1
0.090 (0.609)
0.146 (0.906)
0.145 (0.650)
0.181 (0.727)
0.246 (0.632)
19.1 (3.8)
12.6 (2.6)
17.6 (4.5)
11.9 (3.8)
19.3 (3.8)
1.97 (0.91)
1.32 (0.72)
1.43 (0.68)
1.18 (0.74)
1.91 (0.92)
73.2 (4.4)
63.5 (4.8)
60.1 (0.4)
83.0 (14.2)
I/σ(I)2
3
∆F/σ(∆F)
Anomalous CC4 85.6 (17.6)
(%)
Notes:
1
Rmerge = Σhkl Σi|Iihkl - <Ihkl>|/ Σhkl Σi Iihkl, where I is the intensity of a reflection hkl and <I> is the average
over measurements of hkl.
2
I/σ(I) = < < Ihkl > / σ(<Ihkl >)> where < Ihkl > is the weighted mean of all measurements for a reflection hkl
and σ(<Ihkl >) is the standard deviation of the weighted mean. The values are as reported from SCALA as
Mn(I/sd).
3
∆F/σ(∆F) is average anomalous signal from data truncated to dmin = 4 Å. The values are derived by using
CCP4 programs and are computed as < |ΔF| / σ(ΔF) > where ΔF = |F(h)| - |F(-h)|.
4
Anomalous correlation coefficient evaluated from data truncated to d min = 4 Å.
Values in parentheses are from the highest resolution shell.
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Supplementary Table 2. Crystallographic data collection and refinement statistics
Crystal #1
Data collection
Space group
Unit-cell dimensions
a, b, c (Å)
α, β, γ (°)
Resolution (Å)
Rmerge
I / σ(I)
Completeness (%)
Redundancy
P21
101.42, 101.41, 165,12
90, 101.96, 90
50.00-3.21 (3.27-3.21)*
9.8 (92.3)
11.71 (1.51)
97.9 (83.3)
3.9 (3.5)
Refinement
No. reflections
Rwork / Rfree
No. atoms
Protein
Ligand
Sodium
B-factors
Protein
Ligand
Sodium
R.m.s. deviations
Bond lengths (Å)
Angles (°)
Ramachandran statistics
(%)
Favored
Outliers
52,199
0.2280/0.2913
12390
12263
39
4
98.66
98.42
127.02
66.43
0.010
1.557
88.1
0.2
Notes: *: A single crystal was used for the structure.
Rmerge = Σhkl Σi|Iihkl - <Ihkl>|/ Σhkl Σi Iihkl, where I is the intensity of a reflection hkl and <I> is the average
over measurements of hkl.
Redundancy represents the ratio between the number of measurements and the number of unique reflections.
R factor = Σ|F(obs) – F(cal)|/ΣF(obs); 5% of the data that were excluded from the refinement were used to
calculate Rfree. The average B factor was calculated for all non-hydrogen atoms.
r.m.s.d. of bond is the root-mean-square deviation of the bond angle and length.
Numbers in parentheses are statistics of the highest resolution shell.
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Supplementary Table 3. Distances between Na+ ion and protein coordinating atoms
Distance to Na+ (Å)
Coordinating atoms*
S146 carbonyl O
2.34
S146 hydroxyl O
2.53
S150 carbonyl O
3.33
N151 amide O
2.36
G199 carbonyl O
3.26
Notes: *: Interatomic distances were measured using Chain D.
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Supplementary Fig. 1. Pathway for fatty acid biosynthesis in liver and fat cells. The rate of fatty acid synthesis depends on the cytosolic citrate level. NaCT, Na+-dependent
plasma membrane citrate transporter; TCA, citric acid cycle; CTP, mitochondrial citrate transporter; ACC, acetyl CoA carboxylase; LDL, low-density lipoprotein.
Supplementary Fig. 2. Phylogenetic tree of the DASS family.
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TM1!
TM4b!
HPin!
H4c!
TM5a!
TM5b!
(Supplementary Fig. 3. Continued)
1 0 | W W W. N A T U R E . C O M / N A T U R E
TM3!
TM2!
TM6!
TM4a!
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TM7!
TM8!
TM7!
TM9b!
TM9b!
H9c!
H9c!
TM9a!
TM8!
HPout!
HPout!
TM9a!
TM10a!
TM10a!
TM10b!
TM10b!
TM11!
TM11!
Supplementary Fig. 3. Amino acid sequence alignment of vcINDY and its homologs.
Supplementary
Fig. 3. Amino
acid sequence
alignment
of vcINDY
andcrystal
its homologs.
Positions of secondary
structures
of the protein
as observed
in its
structure are
Positions of secondary structures of the protein as observed in its crystal structure are
indicated, and they are colored using the same rainbow schemes as in Figs. 1-4. The red
indicated,
and they
using for
the carboxyl
same rainbow
as in Figs. 1-4. The red
dots indicate
the are
twocolored
SNT motifs
groupschemes
binding.
dots indicate the two SNT motifs for carboxyl group binding.
W W W. N A T U R E . C O M / N A T U R E | 1 1
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5!
10!
15!
20!
Retention time (min)!
25!
Supplementary Fig. 4. Analytical size-exclusion chromatography trace of purified vcINDY in dodecylmatoside detergent. vcINDY ran as a single,
monodisperse peak at 16.951 mins. The retention times of the monomeric glycerol-3-phosphate transporter (GlpT, 18.654 mins) and the dimeric tetracycline transporter (TetL, 16.133 mins), two membrane transporter proteins with a similar molecular weight as vcINDY, are indicated. 1 2 | W W W. N A T U R E . C O M / N A T U R E
Absorption at 280 nm (a.u.)!
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4 °C!
44 °C!
44 °C!
+ MgCl2!
//!
//!
//!
44 °C!
+ succinate!
44 °C!
+ malate!
44 °C!
+ citrate!
//!
//!
//!
Time (min)!
Supplementary Fig. 5. Effects of various salts on the thermostability of purified vcINDY characterized by analytical size-exclusion chromatography. vcINDY purified in DDM detergent and 100 mM NaCl ran as a sharp peak (without NaCl, the protein would aggregate). Upon being heated to 44 °C for 10 minutes, the peak height dropped by ~50%. The presence of succinate or malate during incubation was able to stabilize the protein. The largest effect in thermostabilization was observed for citrate, in which the peak height was completely recovered, indicating a specific interaction between the transporter and citrate. As a control, MgCl2 had no effect. The void was at ~11 mins. W W W. N A T U R E . C O M / N A T U R E | 1 3
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M51!
M76!
Supplementary Fig. 6. Quality of vcINDY electron density maps.
The 2Fo – Fc map (contoured at 1.5σ) is superimposed with the anomalous difference Fourier map (contoured at 3.7σ) of the SeMet crystal, which was used to identify the position of 22 out of the 23 methionine residues of the vcINDY protein. The crystallographic asymmetric unit cell contains four vcINDY protomers. 1 4 | W W W. N A T U R E . C O M / N A T U R E
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a!
90°!
b!
c!
90°!
Cytosol!
Supplementary Fig. 7. Periplasmic view of the vcINDY dimer. Structure of
the protein is viewed from the extracellular space. The cross section of the protein dimer in the membrane plane measures about 80 Å by 55 Å, whereas the height of the protein along the membrane normal is 60 Å. Many residues at the interface are conserved from bacteria to mammals, including Trp320, which forms an inter-protomer π-π interaction with the Trp320 of the other protomer.
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a!
Cytosol!
90°!
b!
Supplementary Fig. 8. Structure of the vcINDY protomer. a, Viewed from within the membrane. b, Viewed from the cytosol. 1 6 | W W W. N A T U R E . C O M / N A T U R E
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a
b
a
b
TMs 4-6!
TMs
9-11!
TMs
4-6!
TMs 9-11!
90°!
90°!
TMs 7-8!
TMs 7-8!
TMs 4-6!
TMs
9-11!
TMs
4-6!
TMs 9-11!
TMs 2-3!
TMs 2-3!
Supplementary Fig. 9. Comparison of the N-terminal and C-terminal halves
in vcINDY. a.
Overlay
of the N-ofand
with their
Supplementary
Fig.
9. Comparison
theC-terminal
N-terminalhalves,
and C-terminal
helical
bundles
superimposed.
b. Overlay
theC-terminal
N- and C-terminal
helical
halves
in vcINDY.
a. Overlay
of the N-ofand
halves, with
their
bundles.
helical bundles superimposed. b. Overlay of the N- and C-terminal helical bundles. TMs
4-6!
TMs
4-6!
TMs
9-11!
TMs
9-11!
TMs
2-3!
TMs
2-3!
TMs
7-8!
TMs 7-8!
Supplementary
Fig.
10.10.
Comparison
ofof
thethe
N-terminal
and
C-terminal
halves
Supplementary
Fig.
Comparison
N-terminal
and
C-terminal
halves
ofof
thethe
vcINDY
structure.
Overlay
of
the
Nand
C-terminal
halves,
with
their
vcINDY structure. Overlay of the N- and C-terminal halves, with their
thumbs
superimposed.
thumbs
superimposed.
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Na1!
S146!
Supplementary Fig. 11. Electron density (2Fo – Fc map, contoured at 2.5σ
and 3.5σ) for the bound Na+ ion at the Na1 site. The electron density between the hairpin HPin tip and the loop L5ab is too large for a Li+ ion, and its ligand coordination and the conservation of the coordinating amino acid sequence do not support a bound water molecule. Therefore, we infer that the density belongs to a bound Na+ ion. kDa!
160!
80!
50!
20!
Supplementary Fig. 12. Western blot analysis of expression levels of
vcINDY mutants in E. coli for uptake experiments. Most of the mutants
expressed at comparable levels to the wild-type protein, indicating that
any decrease observed in transport activity was not due to reduced
protein expression.
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S377!
S150!
N378!
N151!
G199!
A420!
T373!
S146!
Na1 site (green)
!
!
Na2 site (pink)
!
S146 (Cα) - N151 (s.c.) !
!4.6 Å !
!
!T373 (Cα) to N378 (s.c.) !
!
!6.7 Å
S146 (s.c.) - N151 (s.c.)!
!3.1 Å !
!
!T373 (s.c.) to N378 (s.c.) !
!
!7.6 Å
G199(Cα) - S150 (Cα) !
!6.6 Å !
!
!A420 (Cα) to S377 (Cα) !
!
!8.1 Å
Supplementary Fig. 13. Overlay of Na1 and Na2 site structures and comparison
of distances between the corresponding hairpin tip and capping loop. The distances between the HPout and L10ab in the C-terminal Na2 clamshell (pink) is significantly larger than the corresponding distances between the HPin and L5ab in the N-terminal Na1 clamshell (green), supporting the hypothesis that a Na+ ion has been released from the Na2 site. s.c. denotes side chain.
Supplementary Fig. 14. Electron density (2Fo – Fc map,
contoured at 1.0σ) for the bound citrate molecule. W W W. N A T U R E . C O M / N A T U R E | 1 9
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None!
Succinate!
Sulfate!
1.0!
0.5!
0.0!
WT!
T379P!
Supplementary Fig. 15. Succinate transport activity of wild-type and mutant vcINDY in the presence of sulfate. N = 3. 2 0 | W W W. N A T U R E . C O M / N A T U R E
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a
b
Supplementary Fig. 16. Comparison of the vcINDY protomer structure with that of the concentrative nucleotide transporter (CNT) from Vibrio cholerae. vcINDY is colored orange and yellow, whereas CNT is colored green and cyan. a, Periplasmic view. b, Viewed from within the membrane. The r.m.s.d between Cα atoms of these most conserved parts, the helical hairpins and the two Transmembrane helices that follow them, is 4.9 Å for those 243 amino acid residues. W W W. N A T U R E . C O M / N A T U R E | 2 1
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Co-S-Na+ !
Co !
!
!
!
!
!Ci-S-Na+!
!
Cytosol!
Ci!
Supplementary Fig. 17. Proposed transport mechanism of vcINDY. In the outward-facing Co conformation, the N-terminal half adopts a U-shape, whereas the C-terminal half adopts a V-shape. Following Na+ and substrate binding, the N- and C-terminal halves change to a V- and a U-shape, respectively, resulting in the inward-facing Ci-S-Na+ conformation. After the release of Na+ and substrate to the cytosol, the transporter returns to the Co state, completing a substrate translocation cycle.
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